Process to produce anthracycline glycosides

ABSTRACT

There is presented a process for producing anthracycline glycosides A by converting the sugar moiety of anthracycline glycosides B, represented by the following formula: ##STR1## into the sugar moiety of anthracycline glycosides A, represented by the following formula: ##STR2## with the aid of a microorganism belonging to the species Streptomyces galilaeus or Streptomyces melanogenes.

DESCRIPTION OF THE INVENTION

The present invention relates to a process for the preparation of anthracycline glycosides A.

More particularly, the present invention relates to a process for producing anthracycline glycosides A by converting the sugar moiety of anthracycline glycosides B, represented by the following formula, ##STR3## into the sugar moiety of anthracycline glycosides A, represented by the following formula, ##STR4## with the aid of a microorganism belonging to the species Streptomyces galilaeus or Streptomyces melanogenes.

It is known that anthracycline glycosides A and B are effective against tumours. Furthermore, according to the article, i.e. Umezawa et al., "New antitumor antibiotics, Aclacinomycin A and B", J. Antibiotics, 28,830,1975, it is reported that the antitumor activity of aclacinomycin A is stronger than that of aclacinomycin B. However, according to the conventional fermentation method, there are obtained not only anthracycline glycosides A but also anthracyline glycosides B.

The process provided by the present invention enables anthracycline glycosides A having the stronger antitumor acitivity to be manufactured from anthracycline glycosides B in high yield and in a very simple manner. This process comprises converting the sugar moiety of anthracycline glycosides B, represented by the aforementioned formula B, into the sugar moiety of athracycline glycosides A, represented by the aforementioned formula A, with the air of a microorganism belonging to the species Streptomyces galilaeus or Streptomyces melanogenes, and capable of converting said anthracycline glycoside B into the corresponding anthracycline glycoside A.

The process provided by the present invention conveniently applies to the process for producing especially aclacinomycin A, auramycin A, sulfurmycin A, cinerbin A, 1-hydroxyauramycin A or 1-hydroxysulfurmycin A from each corresponding anthracycline glycoside B.

The chemical structures of said anthracycline glycosides A and B are as follows. ##STR5##

    ______________________________________                                         Anthracycline glycosides                                                                        R.sub.1  R.sub.2                                              ______________________________________                                         Aclacinomycin    H        CH.sub.2 CH.sub.3                                    Auramycin        H        CH.sub.3                                             Sulfurmycin      H        CH.sub.2 COCH.sub.3                                  Cinerubin        OH       CH.sub.2 CH.sub.3                                    1-Hydroxyauramycin                                                                              OH       CH.sub.3                                             1-Hydroxysulfurmycin                                                                            OH       CH.sub.2 COCH.sub.3                                  ______________________________________                                    

The microorganism used in the process provided by the present invention is a microorganism belonging to the species Streptomyces galilaeus or Streptomyces melanogenes and capable of converting anthracycline glycosides B into the corresponding anthracycline glycosides A.

Preferred strains which can be used in the process provided by the present invention are Streptomyces galilaeus OBB-111, Streptomyces galilaeus OBB-731 and Streptomyces galilaeus FR-401 which have been isolated from soils in Neuschwanstein, Oberammergau and Murnau respectively, Oberbayern, West Germany; and Streptomyces galilaeus AC-628 and Streptomyces melanogenes AC-180 which have been isolated from soils in Ohtsu, Kumamoto-ken and Atami, Shizuoka-ken respectively, Japan; as well as mutants and variants thereof, preferably Streptomyces galilaeus OBB-111-848 derived from the strain Streptomyces galilaeus OBB-111 by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. Such mutants can be obtained from the parent strains by conventional mutation methods, for example by irradiation with UV light, X-rays or γ-rays, or by treatment with suitable mutagens. The strains Streptomyces galilaeus OBB-111, Streptomyces galilaeus OBB-731, Streptomyces galilaeus FR-401, Streptomyces galilaeus AC-628, Streptomyces melanogenes AC-180 and Streptomyces galilaeus OBB-111-848 have been deposited in the Agency of Industrial Science and Technology, Fermentation Research Institute, Japan, under FERM-P No. 4780, FERM-P No. 5402, FERM-P No. 4882, FERM-P No. 4783, FERM-P No. 4782 and FERM-P No. 5316 respectively and at the American Type Culture Collection, Rockville, Md., USA under ATCC Nos. 31533, 31615, 31535, 31573, 31572, and 31598 respectively.

The mycological characteristics thereof are as follows:

1. Morphological Properties

The strain OBB-111 (FERM-P No. 4780, ATCC 31533) and the strain OBB-731 (FERM-P No. 5402; ATCC 31615) form moderately long aerial mycelium from substrate mycelium. Hooks or spirals are observed to develop at the apex of the aerial mycelium, but no whorls are formed. Mature spore chains with more than 10 spores per chain are usually produced. The spores are cylindrical, measure 0.5 to 0.6μ×0.3 to 1.0μ and their surface is smooth.

The strain FR-401 (FERM-P No. 4882, ATCC 31535) forms aerial mycelium, branched like tufts, from substrate mycelium. Spirals are observed to develop, but no whorls are formed. Mature spore chains with more than 10 spores per chain are usually produced. The spores are cylindrical or ellipsoidal, mreasure 0.6 to 0.8μ×0.8 to 1.2μ and their surface is smooth.

The strain AC-628 (FERM-P No. 4783; ATCC 31573) developed abundantly aerial mycelia from substrate mycelia. The aerial mycelia formed spirals, but whorl formation was not recognized. Mature spore chain was generally more than 10 spores per chain. The spore was of 0.6˜1.0μ1.0×1.4μ in diameter, cylindrical in shape and its surface was smooth.

The strain AC-180 (FERM-P No. 4782; ATCC 31572) only poorly developed aerial mycelia from substrate mycelia. The aerial mycelia formed were straight to flexuous. Neither spiral nor whorl formation was observed. Mature spore chain was generally more than 10 spores per chain. The spore was of 0.4˜0.6×0.8˜1.2μ in diameter, cylindrical in shape and its surface was smooth.

2. Culture Characteristics on Various Media

The culture characteristics of strains OBB-111, OBB-731, FR-401, AC-628 and AC-180 are shown in Table 1.

3. Physiological Characteristics

The physiological characteristics and carbohydrate utilization of the strains OBB-111, OBB-731, FR-401, AC-628 and AC-180 are shown in Table 2 and Table 3, respectively.

                                      TABLE 1.                                     __________________________________________________________________________     Culture characteristics on various media                                       Medium Strain OBB-111                                                                             Strain FR-401                                                                            Strain AC-628                                                                          Strain OBB-731                                                                              Strain                       __________________________________________________________________________                                                       AC-180                       Sucrose-nitrate                                                                agar                                                                           Growth dull orange [4pe,                                                                          brown [6ni, Taupe                                                                        colorless˜                                                                       pale yellow˜pale                                                                      pink [6gc, Dusty                                                               Coral]                              Orange Rust]                                                                               Brown]˜pale                                                                        pale orange                                                                            yellowish brown                                                                             ˜dull purple                                                             [9le,                                           reddish orange    [3gc, Light Tan]                                                                            Raspberry]                                      [6ie, Redwood]                                              Aerial brownish gray [3cb,                                                                        pinkish white                                                                            light gray                                                                             brownish gray [3cb,                                                                         none                         Mycelium                                                                              Sand]˜pale orange                                                                    [6ge, Rose Gray]                                                                         [2fe, Covert                                                                           Sand]˜pale orange [5cb]                    [5cb]                 Gray]                                             Diffusible                                                                            reddish     reddish   none    yellowish    reddish                      Pigment                                                                        Glucose-asparagine                                                             agar                                                                           Growth dull orange [3pe, Topaz                                                                    pale orange                                                                              yellowish gray                                                                         dull orange [3pe,                                                                           brownish gray [3li,                 ˜3ne, Topaz]                                                                         [4ge, Rose Beige]                                                                        [2ec, Oatmeal]                                                                         Topaz˜3ne, Topaz]                                                                     Beaver]˜dark                                                             yellowish                                                                      brown [3pn, Cepla                                                              Brown]                       Aerial light brownish gray                                                                        none      none    light brownish gray                                                                         none                         Mycelium                                                                              [3dc, Natural]                [3dc, Natural]                            Diffusible                                                                            brownish    brownish  none    brownish     brownish                     Pigment                                                                        Glycerol-asparagine                                                            agar                                                                           Growth pale yellow˜pale                                                                     dull reddish orange                                                                      light reddish                                                                          pale yellow [3gc, Light                                                                     grayish red [7pi, Dark              yellowish brown [3gc,                                                                      [5le, Rust Tan˜5ne                                                                 orange [5pa,                                                                           Tan]˜pale yellowish                                                                   Wine]˜dark                                                               brownish                            Light Tan˜31c, Amber]                                                                Tile Red] Brite Orange]                                                                          brown [3lc, Amber]                                                                          purple [7pn, Dark Rose                                    dull reddish         Brown]                                                    orange [5ne,                                                                   Tile Red]                                         Aerial light gray [2fe,                                                                           light gray [2fe,                                                                         light gray                                                                             light gray [2fe, Covert                                                                     pale pink [7cb, Cloud        Mycelium                                                                              Covert Gray]                                                                               Covert Gray]                                                                             [2fe, Covert                                                                           Gray]        Pink˜7ec, Rose                                                           Mist]                                                     Gray˜3fe,                                                                Silver Gray]                                      Diffusible                                                                            none        brown     reddish yellow       brownish                     Pigment                                                                        Starch-inorganic                                                               salts agar                                                                     (ISP medium No. 4)                                                             Growth pale yellow [2pc, Brite                                                                    grayish yellow [3ec,                                                                     yellowish gray                                                                         pale yellow [2pc, Brite                                                                     light brown [4ng,                                                              Light                               Gold]˜dull yellow                                                                    Bisque]˜pale                                                                       [2ec, Oatmeal]                                                                         Gold]˜dull yellow                                                                     Brown]˜dark                                                              brown                               [2pe, Mustard Gold]                                                                        yellowish brown                                                                          ˜light reddish                                                                   [2pe, Mustard Gold]                                                                         [4pn, Dark Brown]                               [3ge, Lt Tan]                                                                            orange [5pa,                                                                   Brite Orange]                                     Aerial light brownish gray                                                                        light brownish gray                                                                      light gray                                                                             light brownish gray                                                                         pale pink [7ba, Pink         Mycelium                                                                              [2dc, Natural]˜light                                                                 [3dc, Natural]                                                                           [3fe, Covert                                                                           [2dc, Natural]˜light                                                                  Tint]˜pink [7ca,                                                         Pale                                gray [2fe, Covert Gray]                                                                              Gray]   gray [2fe, Covert Gray]                                                                     Pink]                        Diffusible                                                                            yellow      brownish  brownish                                                                               yellow       brown                        Pigment                                                                        Tyrosine agar                                                                  (ISP medium No. 7)                                                             Growth dark brownish gray                                                                         brownish violet                                                                          brown [5ni,                                                                            dark brownish gray                                                                          brown [5ni, Cocoa                   [3ni, Clove Brown]                                                                         [4pe, DK Spice                                                                           Cocoa Brown]                                                                           [3ni, Clove Brown]                                                                          Brown]˜dark                                                              brown                                           Brown]    ˜dull reddish  [5nl, Dark Brown]                                         orange [5le,                                                                   Rust Tan]                                         Aerial none        light gray [3fe,                                                                         light gray                                                                             none         pale pink [7ba, Pink         Mycelium           Silver Gray]                                                                             [3fe, Covert         Tint]                                                     Gray]˜light                                                              brownish gray                                                                  [5fe, Ashes]                                      Diffusible                                                                            black       brown     brown   black        dark brown                   Pigment                                                                        Nutrient agar                                                                  Growth colorless˜pale brown                                                                 colorless˜pale                                                                     pale brown                                                                             colorless˜pale                                                                        pale brown [4ge,                                brown     [3ie, Camel]         Light Fawn]                  Aerial none        none      none    none         none                         Mycelium                                                                       Diffusible                                                                            none        none      none    brown        none                         Pigment                                                                        Yeast extract-malt                                                             extract agar                                                                   (ISP medium No. 2)                                                             Growth yellowish brown                                                                            pale yellowish                                                                           dull red                                                                               yellowish brown                                                                             dull red [7ng, Old                                                             Wine]                               [3ng, Yellow Maple]                                                                        brown [3ie, Camel]                                                                       [5ie, Copper                                                                           [3ng, Yellow Maple]                                                                         ˜dark brownish                                                           purple                                                    Tan]˜reddish   [8pl, Cordvan]                                            brown [5ng,                                                                    Brick Red]                                        Aerial light gray [2fe,                                                                           light gray [2fe,                                                                         light brownish                                                                         light gray [2fe,                                                                            brownish gray [3cb,          Mycelium                                                                              Covert Gray]                                                                               Covert Gray]˜light                                                                 gray [5fe,                                                                             Covert Gray] Sand]                                           brownish gray [3dc,                                                                      Ashes˜7fe,                                                     Natural]  Ashes]                                            Diffusible                                                                            none        none      brownish                                                                               none         brown                        Pigment                                                                        Oatmeal agar                                                                   (ISP medium No. 3)                                                             Growth pale yellowish brown                                                                       pale yellowish                                                                           pale brown                                                                             pale yellowish brown                                                                        dark red [6pi, Brown                [2gc, Bamboo]˜pale                                                                   brown [3ie, Camel]                                                                       [4gc, Nude Tan]                                                                        [2gc, Bamboo]˜pale                                                                    Mahogany]˜dark                                                           brownish                            brown [3ie, Camel]                                                                         ˜pale reddish                                                                      ˜reddish                                                                         brown [3ie, Camel]                                                                          purple [6pn, Dark                                                              Brown                                           brown [4ge,                                                                              brown [6ng,          Mahogany]                                       Nude Tan] Brick Red]                                        Aerial light gray [2fe, Covert                                                                    light grayish                                                                            light gray                                                                             light gray [2fe, Covert                                                                     none                         Mycelium                                                                              Gray˜3fe, Silver Gray]                                                               reddish brown                                                                            [2fe, Covert                                                                           Gray˜3fe, Silver Gray]                                 [5fe, Ashes]                                                                             Gray]˜dark                                                               gray [3ih, Beige                                                               Gray]                                             Diffusible                                                                            brown       red       reddish brown        reddish                      Pigment                                                                        Skimmed milk (37° C.)                                                   Growth brown˜dark brown                                                                     brown˜dark brown                                                                   pale brown                                                                             brown˜dark brown                                                                      colorless˜pale                                                           brown                                                     ˜brown                                      Aerial white˜brownish gray                                                                  none      none    white˜brownish                                                                        none                         Mycelium                                                                       Diffusible                                                                            dark brown  dark brown                                                                               dark brown                                                                             dark brown   none                         Pigment                                                                        Glucose peptone                                                                gelatin stab                                                                   Growth pale yellow colorless colorless˜pale                                                                   pale yellow  colorless˜pale                                      yellowish            yellowish brown                                           brown                                             Aerial none        none      yellowish gray                                                                         none         none                         Mycelium                                                                       Diffusible                                                                            brown       brown     brown   brown        faint brownish               Pigment                                                                        __________________________________________________________________________

                                      TABLE 2.                                     __________________________________________________________________________     Physiological characteristics of the 5 strains                                          OBB-111, OBB-731     Methods and Materials                            Test     FR-401 and AC-6288                                                                        AC-180    Used                                             __________________________________________________________________________     Gelatin  moderate   weak liquefaction                                                                        glucose-peptone-                                 liquefaction                                                                            liquefaction         gelatin medium:                                                                27° C.                                    Starch   weak to moderate                                                                          moderate hydrolysis                                                                      starch-inorganic                                 hydrolysis                                                                              hydrolysis           salts agar                                       Peptonisation                                                                           moderate to strong                                                                        strong coagulation                                                                       10% skimmed milk;                                and coagulation                                                                         peptonisation and                                                                         and weak  37° C.                                    of skimmed milk                                                                         no coagulation                                                                            peptonization                                              Nitrate  positive   negative  ISP medium No. 8;                                reduction                     27° C.                                    Melanin  positive   positive  ISP medium No. 1                                 formation                     ISP medium No. 6                                                               ISP medium No. 7                                 __________________________________________________________________________

                  TABLE 3.                                                         ______________________________________                                         Carbohydrate utilization                                                                  OBB-111, OBB-731                                                               FR-401 and AC-6288                                                                          AC-180                                                 ______________________________________                                         L-Arabinose  positive       positive                                           D-Xylose     positive       positive                                           Glucose      positive       positive                                           D-Fructose   positive       positive                                           Sucrose      positive       positive                                           Inositol     positive       positive                                           L-Rhamnose   positive       positive                                           Raffinose    positive       positive                                           D-Mannitol   negative       positive                                           ______________________________________                                          Basal medium: PridhamGottlieb medium (ISP medium No. 9)                        Temperature: 27° C.                                               

The foregoing characteristics of strains OBB-111, OBB-731, AC-628 and FR-401 can be summarised as follows: The strains belong to the genus Streptomyces. The aerial mycelium forms spirals at the apex but no whorls. The surface of the spores is smooth. The growth on various media is found to be pale yellowish brown to pale brown or dull orange, and the aerial mycelium is light grey. The strains produce reddish to brown diffusible pigment and melanin on various media. Among known species of Streptomyces, strains OBB-111, OBB-731, AC-628 and FR-401 resemble Streptomyces galilaeus (Reference 1: Archiv fur Mikrobiologie, 31, 356, 1958. Reference 2: The Actinomycetes, 2, 215, 1961. Reference 3: International Journal of Systematic Bacteriology, 22, 298, 1972) and Streptomyces galilaeus MA 144-M1, FERM-P No. 2455 (Reference 1: Japanese Patent Publication No. 34915/1976). The differences between the present strains and the standard strains of S. galilaeus ISP 5481 and S. galilaeus MA 144-M1 (FERM-P No. 2455) were investigated by parallel cultures. The results are shown in Table 4 hereinafter.

                  TABLE 4                                                          ______________________________________                                                    OBB-111,          S. galilaeus                                                 OBB-731,          MA 144-M1                                                    FR-401 and                                                                             S. galilaeus                                                                             (FERM-P No.                                                  AC-628  ISP 5481  2455)                                             ______________________________________                                         Liquefaction of                                                                             moderate  weak to   weak to                                       gelatin                moderate  moderate                                      Coagulation of                                                                              negative  weak      negative                                      milk                   positive                                                Diffusible   dark brown                                                                               light brown                                                                              dark brown                                    pigment                                                                        Change of colour                                                               of growth by 0.05N                                                             sodium hydroxide                                                               solution:                                                                      ISP medium No. 3                                                                            pink to    --       pink to                                                    violet              violet                                        ISP medium No. 4                                                                            slight pink                                                                               --       slight pink                                                ˜violet       ˜violet                                 ISP medium No. 5                                                                            violet    slight    violet                                                               violet                                                  ______________________________________                                    

From the results, the present strains are very similar to S. galilaeus ISP 5481 and S. galilaeus MA 144-M1 (FERM-P No. 2455) in morphology and colour of the growth and mycelium on various media, chromogenicity and utilization of carbohydrates, although some differences were observed. We, therefore, concluded that OBB-111 (FERM-P No. 4780), OBB-731 (FERM-P No. 5402), FR-401 (FERM-P No. 4782) and AC-628 (FERM-P No. 4783) belong to a species of Streptomyces galilaeus and named Streptomyces galilaeus OBB-111, OBB-731, FR-401 and AC-628, respectively.

On the other hand, strain AC-180 (FERM-P No. 4782), belonging to genus Streptomyces, developed straight to flexuous aerial mycelia, though poorly. The spore surface was smooth. Growth was dark brown to dark brownish purple and developed poorly aerial mycelia of pale pink on ISP Medium 2, 4, 5 and 7, however no aerial mycelium was formed on sucrose nitrate agar, glucose asparagine agar, nutrient agar and ISP Medium 3. Reddish to brownish pigment was produced in various agar media. Melanoid pigment was produced in ISP-Medium 1, 6 and 7. In carbon utilization test, abundant growth was seen with L-arabinose, D-xylose, glucose, D-fructose, inositol, L-rhamnose, raffinose and D-mannitol. Sucrose seemed probably utilized. With skimmed milk test, both coagulation and peptonization were observed. Starch hydrolysis and gelatin liquefaction were positive, while nitrate was not reduced to nitrite.

When all the above properties were compared with known species of Streptomyces, it was found that many features of AC-180 (FERM-P No. 4782) were common with strain Streptomyces malanogenes (Int. J. Syst. Bact., 18, p. 348, 1968; J. Antibiot., Tokyo Ser. A, 10, 138˜142, 1957). Thus a comparative study was undertaken with Streptomyces melanogenes IFO 12890 by parallel culture, the results of which are summarized in Table 5. The strain AC-180 (FERM-P No. 4782) differed from Streptomyces melanogenes IFO 12890 strain in L-rhamnose utilization and the color of reverse side (AC-180 (FERM-P No. 4782) was purplish color strong) on ISP-Medium 5. However, these differences were small in balance with other great similarities in morphology cultural and physiological properties. Thus, we concluded that strain AC-180 (FERM-P No. 4782) should be reasonably a species of Streptomyces melanogenes and identified Streptomyces melanogenes AC-180.

                  TABLE 5.                                                         ______________________________________                                         Comparison of strain AC-180                                                    with Streptomyces melanogenes IFO 12890                                                     AC-180      St. melanogenes                                                    (FERM-P No. 4782)                                                                          IFO 12890                                             ______________________________________                                         Morphology of aerial                                                                          straight to flexuous                                                                         straight to                                       mycelium                     flexuous                                          Color of aerial mycelium                                                                      red to gray color                                                                            red to gray                                                      series        color series                                      Condition of aerial                                                                           none or scant moderate                                          mycelium                                                                       Reverse side color                                                                            dark brown to dark                                                                           brown to dark                                     (on ISP-Medium 5)                                                                             brownish purple                                                                              brown                                             Soluble pigment                                                                               reddish to brownish                                                                          brownish                                          Skimmed milk                                                                   Coagulation    +             +                                                 Peptonization  +             +                                                 Gelatin liquefaction                                                                          +             +                                                 Nitrate reduction                                                                             -             -                                                 Carbon utilization                                                             L-arabinose    ++            ++                                                D-xylose       ++            ++                                                Glucose        ++            ++                                                D-fructose     ++            ++                                                Sucrose        +             +                                                 Inositol       ++            ++                                                L-rhamnose     ++            -                                                 Raffinose      ++            ++                                                D-mannitol     ++            ++                                                ______________________________________                                    

Streptomyces galilaeus OBB-111-848 (FERM-P No. 5316; ATCC 31598) is the strain derived from Streptomyces galilaeus OBB-111 (FERM-P No. 4780, ATCC 31533) by the following method.

The spores of an agar slant culture of Streptomyces galilaeus OBB-111 (FERM-P No. 4780, ATCC 31533) were suspended in 10 ml of sterile physiological saline solution and filtered through a glass filter No. 3. The spores were resuspended and diluted 2-fold with 0.2 M tris buffer (pH 9.0) containing 2 mg/ml of N-methyl-N'-nitro-N-nitrosoguanidine and incubated at 27° C. for 60 minutes. Then the spores were collected on the Nucleopore filter (0.2 μm pore size), washed with 30 ml of sterile physiological saline solution and resuspended in 10 ml of sterile physiological saline solution. The spore suspension thus obtained was spread on the ISP-No. 2 medium in a Petri dish and incubated at 27° C. for 4˜6 days. The colonies were picked up and transferred to an agar slant and incubated for 10˜14 days. The strains thus obtained were tested for production of auramycinone and sulfurmycinone glycosides according to the following procedure.

The spores of each strain was inoculated into 10 ml of the medium in a test tube consisting of 2% glucose, 2% soluble starch 1% Pharmadmeia (Traders Oil Mill Co., U.S.A.), 0.1% K₂ HPO₄, 0.1% MgSO₄.7H₂ O, 0.3% NaCl and 0.3% CaCO₃ and cultured for 4 days at 27° C. on a reciprocal shaker. The culture broth was extracted with 20 ml of a solvent mixture of chloroform and methanol (1:1, v/v) and chloroform layer recovered. After concentration to dryness, the extract was dissolved in 0.5 ml of chloroform and analyzed on silica gel tlc plates developed with toluene/methanol (10:1).

Streptomyces galilaeus OBB-111-848 (FERM-P No. 5316; ATCC 31598) was selected as a non-producer of anthracycline pigments.

The microorganism can be used in a form of the culture broth, mycelia or enzyme. The culture broth can be prepared by inoculating a suitable medium with the microorganism. The culture medium can contain carbon sources, nitrogen sources, inorganic salts and other nutrient substances suitable for the growth of the microorganisms. Preferred carbon sources are glucose, sucrose, dextrin, mannose, starch, lactose, glycerol and the like. The nitrogen sources are, for example, nitrogen-containing organic substances such as peptone, meat extract, yeast extract, corn steep liquor, casein and the like, or nitrogen-containing inorganic compounds such as nitrates and inorganic ammonium salts. Examples of inorganic salts are phosphates or sodium, potassium, manganese, magnesium, iron copper salts and the like.

The cultivation of the microorganism may be carried out as a submerged culture, as a shaking culture or as a stationary culture. In a preferred embodiment, the microorganism is cultured under aerobic conditions.

The process provided by the present invention is conveniently carried out by adding an anthracycline glycoside B to the cultivated culture broth. The concentration of the anthracycline glycoside B conveniently is up to 1 g/l. The corresponding anthracycline glycoside A can be obtained by continuing the incubation of the microorganism under the aforementioned conditions. The incubation time can vary depending on the microorganism used, on the composition of the culture medium, on the anthracycline glycoside B used and on its concentration. However, in general, an incubation time of several hours suffices. The cultivation temperature generally lies between 20° C. and 30° C. Furthermore, the cultivation is conveniently carried out at a pH of 5 to 7.

The anthracycline glycisode B can be added to the culture of the microorganism during the cultivation or to the culture medium prior to sterilization or inoculation.

The transformation product can be isolated from the fermentation mixture in a manner known per se; for example, by solvent extraction with an organic solvent such as chloroform, methanol, and the like, and/or by chromatography on a carrier such as silica gel, aluminium oxide and the like. The product can be also purified by recrystalization from an organic solvent.

According to another aspect of preferred embodiment of the process provided by the present invention, the reaction can be carried out in the presence of the mycelium isolated from the culture broth of the microorganism or an enzyme extract prepared in a manner known per se from the culture broth or the mycelium, in a solution, for example, a buffer solution, in physiological salt solution, in fresh nutrient solution or in water.

The following Examples illustrate the present invention:

EXAMPLE 1

100 ml of sterilized medium in a 500 ml Erlenmeyer flask were inoculated with a slant of Streptomyces galilaeus OBB-111-848 (FERM-P No. 5316, ATCC 31598). The composition of said medium is as follows: glucose 2%, soluble starch 2%, Pharma Media (Traders Oil Mill Co., U.S.A.) 1% K₂ HPO₄ 0.1%, MgSO₄.7H₂ O 0.1%, NaCl 0.3% and CaCO₃ 0.3%. The flask was incubated at 27° C. for 3 days on a rotary shaker operating at 180 rpm.

2 ml of the culture thus obtained were seeded into each of three 500 ml flasks containing 100 ml of medium with the same composition as mentioned above. After cultivation at 27° C. for 3 days, to each flask were added 5 mg of aclacinomycin B, auramycin B or sulfurmycin B dissolved in 2 ml of methanol and the flasks were further incubated. After 1, 3 and 7 hours, 5 ml aliquots were removed from each flask and extracted with chloroform/methanol (1:1, v/v); and the extract was analyzed on thin layer chromatographic plates developed with a solvent mixture of toluene and methanol (10:1, v/v).

The results are shown in Table 6

                  TABLE 6                                                          ______________________________________                                         Reaction                                                                              Products {(A/A + B) × 100}                                        time   Aclacinomycin A                                                                              Auramycin A                                                                               Sulfurmycin A                                  ______________________________________                                         1 hr.   22.5%         20.4%      23.3%                                         3 hr.  47.8          40.7       40.0                                           7 hr.  63.2          75.0       81.3                                           ______________________________________                                    

EXAMPLE 2

100 ml of sterilized medium in a 500 ml Erlenmeyer flask were inoculated with a slant culture of Streptomyces galilaeus OBB-111-848 (FERM-P No. 5316, ATCC 31598). The composition of said medium is as follows: glucose 2%, soluble starch 2%, Pharma Media (Traders Oil Mill Co., U.S.A.) 1%, K₂ HPO₄ 0.1%, MgSO₄.7H₂ O 0.1%, NaCl 0.3% and CaCO₃ 0.3%. The flask was incubated at 27° C. for 3 days on a rotary shaker operating at 180 rpm.

5 ml of the culture thus obtained were transferred into each of three 500 ml flasks containing 100 ml of medium with the same composition as mentioned above. After cultivation at 27° C. for 2 days, the cells were collected by centrifugation and distributed into three 500 ml flasks containing 5 mg of aclacinomycin B, auramycin B or sulfurmycin B in 100 ml of sterilized saline solution. The flasks were incubated at 27° C. on a rotary shaker.

After 1, 3 and 7 hours, 5 ml aliquots were removed and extracted with chloroform/methanol (1:1, v/v); and the extract was analyzed by high pressure liquid chromatography.

The results thereof are shown in Table 7.

                  TABLE 7                                                          ______________________________________                                         Reaction                                                                              Products (A/A + B) × 100                                          time   Aclacinomycin A                                                                              Auramycin A                                                                               Sulfurmycin A                                  ______________________________________                                         1 hr.   35.5%         38.2%      41.7%                                         3 hr.  48.8          52.9       55.8                                           7 hr.  58.8          65.0       68.2                                           ______________________________________                                    

EXAMPLE 3

In a manner analogous to that described in Example 2, using cinerubin B, 1-hydroxyauramycin B and 1-hydroxysulfurmycin B respectively, there were obtained the conversion ratios of 40%, 43% and 45%, respectively.

EXAMPLE 4

In a manner analogous to that described in Example 2, using Streptomyces galilaeus FR-401 (FERM-P No.4882; ATCC 31535) instead of the strain OBB-111-848, the conversion ratio of aclacinomycin B into aclacinomycin A was 79% after 5 hours.

EXAMPLE 5

In a manner analogous to that described in Example 2, using Streptomyces galilaeus AC-628 (FERM-P No. 4783; ATCC 31573) and Streptomyces melanogenes AC-180 (FERM-P No. 4782, ATCC 31572) respectively, instead of the strain OBB-111-848, the conversion ratios of cinerubin B into cinerubin A after 5 hours were 10% and 30%, respectively.

EXAMPLE 6

In a manner analogous to that described in Example 2, using Streptomyces galilaeus OBB-111 (FERM-P No. 4780, ATCC 31533) instead of Streptomyces galilaeus OBB-111-848, the conversion ratios of aclacinomycin B, auramycin B and sulfurmycin B into aclacinomycin A, auramycin A and sulfurmycin A after 7 hours were 64%, 69% and 73%, respectively.

EXAMPLE 7

The slant culture of Streptomyces galilaeus OBB-111-848 (FERM-P No. 5316, ATCC 31598) was inoculated into 100 ml of medium containing glucose 2%, soluble starch 2%, Pharma Media (Traders Oil Co., U.S.A.) 1%, K₂ HPO₄ 0.1%, MgSO₄.7H₂ O 0.1%, NaCl 0.3%, and CaCO₃ 0.3% in a 500 ml Erlenmeyer flask and cultivated at 27° C. for 3 days on a rotary shaker operating at 180 rpm. 5 ml of the culture thus obtained were transferred into each of fifteen 500 ml Erlenmeyer flasks containing 100 ml of medium with the same composition as mentioned above and incubated for further 2 days under the same condition.

The culture was centrifuged at 8,000 rpm to harvest the cells. The cells were distributed into fifteen flasks containing 100 ml of sterilized saline solution with 5 mg of sulfurmycin B. The flasks were incubated at 27° C. on a rotary shaker at 180 rpm for 7 hours.

The reaction mixture was centrifuged at 8,000 rpm for 10 minutes to separate the cells from the supernatant. The cells were extracted with 1 l of the solvent mixture of chloroform and methanol (1:1, v/v), to which 500 ml of water were added to separate chloroform layer. On the other hand, the supernatant was extracted with the equal volume of chloroform. The extracts were combined and evaporated to dryness and dissolved in toluene, to which was added silica gel. The suspension was shaken overnight at room temperature.

The silica gel was collected by filtration and extracted with chloroform/methanol (5:1, v/v). The extract was evaporated to dryness under reduced pressure and dissolved in a small amount of chloroform. The purification was performed by thin layer chromatography on silica gel 60, F₂₅₄ (Merck Co.) with the solvent system toluene/methanol (10:1), whereby 39 mg of sulfurmycin A were obtained as yellow powder.

EXAMPLE 8

In a manner analogous to that described in Example 7, using auramycin B instead of sulfurmycin B, there were obtained 39 mg of auramycin A as yellow powder.

EXAMPLE 9

In a manner analogous to that described in Example 7, using Streptomyces galilaeus OBB-111-848 and aclacinomycin B instead of sulfurmycin B, there was obtained 50 mg of aclacinomycin A. 

What is claimed:
 1. A process for producing anthracycline glycosides A which comprises converting the sugar moiety of anthracycline glycosides B of the formula: ##STR6## into the sugar moiety of anthracycline glycosides of the formula: ##STR7## by fermentative reaction with a microorganism of the species Streptomyces galilaeus or Streptomyces melanogenes.
 2. The process of claim 1, wherein the microorganism is selected from the group consisting of Streptomyces galilaeus OBB-111 (FERM-P No. 4780; ATCC 31533), Streptomyces galilaeus OBB-731 (FERM-P No. 5402; ATCC 31615), Streptomyces galilaeus FR-401 (FERM-P No. 4882; ATCC 31535), Streptomyces galilaeus AC-628 (FERM-P No. 4783; ATCC 31573), Streptomyces melanogenes AC-180 (FERM-P No. 4782; ATCC 31572) and Streptomyces galilaeus OBB-111-848 (FERM-P No. 5316; ATCC 31598).
 3. The process of claim 1, wherein the anthracycline glycoside B is selected from the group consisting of aclacinomycin B, auramycin B, sulfurmycin B, cinerubin B, 1-hydroxyauramycin B or 1-hydroxysulfurmycin B.
 4. The process of claim 1, wherein the anthracycline glycoside A is selected from the group consisting of aclacinomycin A, auramycin A, sulfurmycin A, cinerubin A, 1-hydroxyauramycin A or 1-hydroxysulfurmycin A.
 5. The process of claim 1, wherein the anthracycline glycosides B are fermented in a culture medium and the corresponding anthracycline glycosides A are recovered from the fermentation broth.
 6. The process of claim 5, wherein the substrate concentration is up to 1 g/l.
 7. The process of claim 1 wherein the reaction is carried out in the presence of the mycelium isolated from the culture broth of the microorganism in a solution. 